Superparamagnetic iron oxide nanoparticles (SPIONs) offer various possibilities for application in medicine. Comprehensive toxicological investigations based on proper physicochemical nanoparticle characterizations are a prerequisite for translation from bench to bedside. Since nanoparticles are prone to interfere with several classical toxicological readout systems, alternative methods are necessary. Multiparameter stainings for flow cytometry and fluorescence microscopy, label-free real-time cell analyses, 3D cell culture, and flow models will be discussed here.